The scientific problem is to find small molecule inhibitors of pancreatic ductal adenocarcinoma (PDAC). The approach follows the observation that people with type 2 or gestational diabetes are at increased risk for PDAC. The hypothesis is that pancreatic progenitor cells activated by sustained periods of high insulin demand are critical to the formation of PDAC in people with diabetes who are genetically predisposed to cancer. However, the contribution of pancreatic progenitor cells to beta cell expansion and PDAC is controversial and has been hampered by challenges in identifying and characterizing this unique cell population. Extensive preliminary data indicates that expression of Rgs16, a regulator of G protein signaling, marks stimulated pancreatic progenitor cells. The innovative approach uses Rgs gene expression to identify ligands of G protein coupled receptors (GPCRs) that participate in the initiation and progression of PDAC. To achieve these advances, focused libraries of putative GPCR ligands will be screened to identify small synthetic molecules that regulate Rgs16 expression in PDAC cell culture. RNA-Seq analysis shows these cells express markers of embryonic progenitors of the ductal and endocrine lineage. Hits will be validated in cell culture and in vivo for effects on Rgs16 expression and progenitor cell biology. The proposed screen has four crucial advantages; appropriate cell types to be tested, facile cell culture assays, the diverse library of rationally chosen compounds, and mouse models for in vivo validation. The assay system uses PDAC cells obtained from transgenic mice that express green fluorescent protein (eGFP) from the Rgs16 gene. Importantly, the Rgs:GFP reporter gene is regulated by GPCR ligands. Pancreatic progenitor cells express GFP as they first differentiate in embryogenesis, indicating they respond to GPCR ligands. The GFP reporter is undetectable in normal adults but is reactivated in the earliest preneoplastic lesions in the pancreas, and maintained throughout PDAC tumor progression. The GPCRs that are expressed in Rgs16:GFP+ PDAC cancer progenitor cells, embryonic and normal adult pancreas were identified among all GPCRs in the mouse genome. A putative agonist for one of these GPCRs, an isoxazole derivative (ISX), induces Rgs16:GFP in PDAC culture. ISX serves as a positive control and proof of principle for the screen. Aim 1 will identify candidate molecules that regulat Rgs16 expression and characterize the effect of ISX and other 'hit' compounds on PDAC cell biology, including the induction or maintenance of a progenitor cell phenotype. Aim 2 will determine the in vivo effects of ISX and other 'hit' compounds on PDAC progression. The approach is innovative because it pioneers using Rgs gene expression to identify GPCRs and their ligands and then tests their function in cell culture and in vivo. The proposed research is significant because successful completion of these aims will lead to major advances in pancreatic cancer surveillance and identify putative druggable targets for therapeutic intervention.